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Background: The KDM1A histone demethylase regulates the cellular balance between proliferation and differentiation, and is often deregulated in human cancers including the childhood tumor neuroblastoma. We previously showed that KDM1A is strongly expressed in undifferentiated neuroblastomas and correlates with poor patient prognosis, suggesting a possible clinical benefit from targeting KDM1A. Methods: Here, we tested the efficacy of NCL-1, a small molecule specifically inhibiting KDM1A in preclinical models for neuroblastoma. Results: NCL-1 mimicked the effects of siRNA-mediated KDM1A knockdown and effectively inhibited KDM1A activity in four neuroblastoma cell lines and a patient-representative cell model. KDM1A inhibition shifted the aggressive tumor cell phenotypes towards less aggressive phenotypes. The proliferation and cell viability was reduced, accompanied by the induction of markers of neuronal differentiation. Interventional NCL-1 treatment of nude mice harboring established neuroblastoma xenograft tumors reduced tumor growth and inhibited cell proliferation. Reduced vessel density and defects in blood vessel construction also resulted, and NCL-1 inhibited the growth and tube formation of HUVEC-C cells in vitro. Conclusions: Inhibiting KDM1A could attack aggressive neuroblastomas two-fold, by re-directing tumor cells toward a less aggressive, slower-growing phenotype and by preventing or reducing the vascular support of large tumors. [ABSTRACT FROM AUTHOR]

Simple Summary: The invasive nature of surgical biopsies prevents their sequential application to monitor disease. Single biopsies fail to reflect cancer dynamics, intratumor heterogeneity, and drug sensitivities that change over time. Detection and characterization of cell-free circulating tumor DNA in biofluids from patients with solid tumors may better support disease monitoring and provide advanced molecular information for clinical decision-making toward personalized medicine. Here, we investigated the cell-free DNA characteristics in blood, bone marrow, cerebrospinal fluid, and urine provided from 84 infants and children with low-, intermediate-, or high-risk neuroblastoma. We report characteristic size distribution and concentration patterns for each biofluid to provide information to support the development of successful liquid biopsy biobanking strategies. We investigate potential correlations between disease activity and cfDNA concentration and provide strong evidence that markers specific for neuroblastoma can be detected in very small blood volumes from infants. Liquid biopsy strategies in pediatric patients are challenging due to low body weight. This study investigated cfDNA size distribution and concentration in blood, bone marrow, cerebrospinal fluid, and urine from 84 patients with neuroblastoma classified as low (n = 28), intermediate (n = 6), or high risk (n = 50) to provide key data for liquid biopsy biobanking strategies. The average volume of blood and bone marrow plasma provided ranged between 1 and 2 mL. Analysis of 637 DNA electropherograms obtained by Agilent TapeStation measurement revealed five different major profiles and characteristic DNA size distribution patterns for each of the biofluids. The proportion of samples containing primarily cfDNA was, at 85.5%, the highest for blood plasma. The median cfDNA concentration amounted to 6.28 ng/mL (blood plasma), 58.2 ng/mL (bone marrow plasma), 0.08 ng/mL (cerebrospinal fluid), and 0.49 ng/mL (urine) in samples. Meta-analysis of the dataset demonstrated that multiple cfDNA-based assays employing the same biofluid sample optimally require sampling volumes of 1 mL for blood and bone marrow plasma, 2 mL for cerebrospinal fluid, and as large as possible for urine samples. A favorable response to treatment was associated with a rapid decrease in blood-based cfDNA concentration in patients with high-risk neuroblastoma. Blood-based cfDNA concentration was not sufficient as a single parameter to indicate high-risk disease recurrence. We provide proof of concept that monitoring neuroblastoma-specific markers in very small blood volumes from infants is feasible. [ABSTRACT FROM AUTHOR]

Let Sg be the orientable surface of genus g and denote by 𝒮g(n,m) the class of all graphs on vertex set [n]={1,...,n} with m edges embeddable on Sg. We prove that the component structure of a graph chosen uniformly at random from 𝒮g(n,m) features two phase transitions. The first phase transition mirrors the classical phase transition in the Erdős‐Rényi random graph G(n,m) chosen uniformly at random from all graphs with vertex set [n] and m edges. It takes place at m=n2+O(n2/3), when the giant component emerges. The second phase transition occurs at m=n+O(n3/5), when the giant component covers almost all vertices of the graph. This kind of phenomenon is strikingly different from G(n,m) and has only been observed for graphs on surfaces. [ABSTRACT FROM AUTHOR]

We consider k‐dimensional random simplicial complexes generated from the binomial random (k + 1)‐uniform hypergraph by taking the downward‐closure. For 1 ≤ j ≤ k − 1, we determine when all cohomology groups with coefficients in F2 from dimension one up to j vanish and the zero‐th cohomology group is isomorphic to F2. This property is not deterministically monotone for this model, but nevertheless we show that it has a single sharp threshold. Moreover we prove a hitting time result, relating the vanishing of these cohomology groups to the disappearance of the last minimal obstruction. We also study the asymptotic distribution of the dimension of the j‐th cohomology group inside the critical window. As a corollary, we deduce a hitting time result for a different model of random simplicial complexes introduced by Linial and Meshulam, previously only known for dimension two. [ABSTRACT FROM AUTHOR]

Accurate modeling of intratumor heterogeneity presents a bottleneck against drug testing. Flexibility in a preclinical platform is also desirable to support assessment of different endpoints. We established the model system, OHC‐NB1, from a bone marrow metastasis from a patient diagnosed with MYCN‐amplified neuroblastoma and performed whole‐exome sequencing on the source metastasis and the different models and passages during model development (monolayer cell line, 3D spheroid culture and subcutaneous xenograft tumors propagated in mice). OHC‐NB1 harbors a MYCN amplification in double minutes, 1p deletion, 17q gain and diploid karyotype, which persisted in all models. A total of 80–540 single‐nucleotide variants (SNVs) was detected in each sample, and comparisons between the source metastasis and models identified 34 of 80 somatic SNVs to be propagated in the models. Clonal reconstruction using the combined copy number and SNV data revealed marked clonal heterogeneity in the originating metastasis, with four clones being reflected in the model systems. The set of OHC‐NB1 models represents 43% of somatic SNVs and 23% of the cellularity in the originating metastasis with varying clonal compositions, indicating that heterogeneity is partially preserved in our model system. What's new? Intratumor heterogeneity is becoming an accepted feature of high‐risk tumors but models reliably capturing this feature remain elusive. Here the authors developed in vitro and in vivo neuroblastoma models from a single aspirate of a neuroblastoma bone marrow metastasis and analyzed the germline patient background, the metastasis and the models via whole‐exome sequencing. The new OHC‐NB1 model system partly reflects the genetic and clonal heterogeneity of the initial biopsy and presents an important first step towards accurate preclinical modeling of neuroblastoma. [ABSTRACT FROM AUTHOR]

For integers g;m 0 and n > 0, let Sg(n;m) denote the graph taken uniformly at random from the set of all graphs on f1; 2; : : : ; ng with exactly m = m(n) edges and with genus at most g. We use counting arguments to investigate the components, subgraphs, maximum degree, and largest face size of Sg(n;m), finding that there is often difeerent asymptotic behavior depending on the ratio m n . In our main results, we show that the probability that Sg(n;m) contains any given nonplanar component converges to 0 as n ! 1 for all m(n), the probability that Sg(n;m) contains a copy of any given planar graph converges to 1 as n ! 1 if lim inf m n > 1, the maximum degree of Sg(n;m) is (ln n) with high probability if lim inf m n > 1, and the largest face size of Sg(n;m) has a threshold around m n = 1, where it changes from (n) to (ln n) with high probability. [ABSTRACT FROM AUTHOR]

LIN28B regulates developmental processes by modulating microRNAs (miRNAs) of the let-7 family. A role for LIN28B in cancer has been proposed but has not been established in vivo. Here, we report that LIN28B showed genomic aberrations and extensive overexpression in high-risk neuroblastoma compared to several other tumor entities and normal tissues. High LIN28B expression was an independent risk factor for adverse outcome in neuroblastoma. LIN28B signaled through repression of the let-7 miRNAs and consequently resulted in elevated MYCN protein expression in neuroblastoma cells. LIN28B-let-7-MYCN signaling blocked differentiation of normal neuroblasts and neuroblastoma cells. These findings were fully recapitulated in a mouse model in which LIN28B expression in the sympathetic adrenergic lineage induced development of neuroblastomas marked by low let-7 miRNA levels and high MYCN protein expression. Interference with this pathway might offer therapeutic perspectives. [ABSTRACT FROM AUTHOR]

Given a graph G, the genus of G denotes the smallest integer g for which G can be drawn on the orientable surface of genus g without crossing edges. For integers g;m 0 and n > 0, we let Sg(n;m) denote the graph taken uniformly at random from the set of all graphs on f1. 2....ng with exactly m = m(n) edges and with genus at most g = g(n). We investigate the evolution of Sg(n;m) as m increases, focussing on the number |H1| of vertices in the largest component. For g<

We consider a model of a random simplicial complex generated by taking the downward-closure of a non-uniform binomial random hypergraph, in which each set of k + 1 vertices forms an edge with some probability pk independently, where pk depends on k and on the number of vertices n. We consider a notion of connectedness on this model according to the vanishing of cohomology groups over an arbitrary abelian group R. We prove that this notion of connectedness displays a phase transition and determine the threshold. We also prove a hitting time result for a natural process interpretation, in which simplices and their downward-closure are added one by one. [ABSTRACT FROM AUTHOR]

Purpose: Treating refractory or relapsed neuroblastoma remains challenging. Monitoring body fluids for tumor-derived molecular information indicating minimal residual disease supports more frequent diagnostic surveillance and may have the power to detect resistant subclones before they give rise to relapses. If actionable targets are identified from liquid biopsies, targeted treatment options can be considered earlier., Experimental Design: Droplet digital PCR assays assessing MYCN and ALK copy numbers and allelic frequencies of ALK p.F1174L and ALK p.R1275Q mutations were applied to longitudinally collected liquid biopsies and matched tumor tissue samples from 31 patients with high-risk neuroblastoma. Total cell-free DNA (cfDNA) levels and marker detection were compared with data from routine clinical diagnostics., Results: Total cfDNA concentrations in blood plasma from patients with high-risk neuroblastoma were higher than in healthy controls and consistently correlated with neuron-specific enolase levels and lactate dehydrogenase activity but not with 123I-meta-iodobenzylguanidine scores at relapse diagnosis. Targeted cfDNA diagnostics proved superior for early relapse detection to all current diagnostics in 2 patients. Marker analysis in cfDNA indicated intratumor heterogeneity for cell clones harboring MYCN amplifications and druggable ALK alterations that were not detectable in matched tumor tissue samples in 17 patients from our cohort. Proof of concept is provided for molecular target detection in cerebrospinal fluid from patients with isolated central nervous system relapses., Conclusions: Tumor-specific alterations can be identified and monitored during disease course in liquid biopsies from pediatric patients with high-risk neuroblastoma. This approach to cfDNA surveillance warrants further prospective validation and exploitation for diagnostic purposes and to guide therapeutic decisions., (©2022 American Association for Cancer Research.)

The invasive nature of surgical biopsies deters sequential application, and single biopsies often fail to reflect tumor dynamics, intratumor heterogeneity and drug sensitivities likely to change during tumor evolution and treatment. Implementing molecular characterization of cell-free neuroblastoma-derived DNA isolated from blood plasma could improve disease assessment for treatment selection and monitoring of patients with high-risk neuroblastoma. We established droplet digital PCR (ddPCR) protocols for MYCN and ALK copy number status in plasma from neuroblastoma patients. Our ddPCR protocol accurately discriminated between MYCN and ALK amplification, gain and normal diploid status in a large panel of neuroblastoma cell lines, and discrepancies with reported MYCN and ALK status were detected, including a high-level MYCN amplification in NB-1, a MYCN gain in SH-SY5Y, a high-level ALK amplification in IMR-32 and ALK gains in BE(2)-C, Kelly, SH-SY5Y and LAN-6. MYCN and ALK status were also reliably determined from cell-free DNA derived from medium conditioned by the cell lines. MYCN and ALK copy numbers of subcutaneous neuroblastoma xenograft tumors were accurately determined from cell-free DNA in the mouse blood plasma. In a final validation step, we accurately distinguished MYCN and ALK copy numbers of the corresponding primary tumors using retrospectively collected blood plasma samples from 10 neuroblastoma patients. Our data justify the further development of molecular disease characterization using cell-free DNA in blood plasma from patients with neuroblastoma. This expanded molecular diagnostic palette may improve monitoring of disease progression including relapse and metastatic events as well as therapy success or failure in high-risk neuroblastoma patients., Competing Interests: CONFLICTS OF INTEREST None.

Polo-like kinase 1 (PLK1) is a serine/threonine kinase that promotes G2/M-phase transition, is expressed in elevated levels in high-risk neuroblastomas and correlates with unfavorable patient outcome. Recently, we and others have presented PLK1 as a potential drug target for neuroblastoma, and reported that the BI2536 PLK1 inhibitor showed antitumoral actvity in preclinical neuroblastoma models. Here we analyzed the effects of GSK461364, a competitive inhibitor for ATP binding to PLK1, on typical tumorigenic properties of preclinical in vitro and in vivo neuroblastoma models. GSK461364 treatment of neuroblastoma cell lines reduced cell viability and proliferative capacity, caused cell cycle arrest and massively induced apoptosis. These phenotypic consequences were induced by treatment in the low-dose nanomolar range, and were independent of MYCN copy number status. GSK461364 treatment strongly delayed established xenograft tumor growth in nude mice, and significantly increased survival time in the treatment group. These preclinical findings indicate PLK1 inhibitors may be effective for patients with high-risk or relapsed neuroblastomas with upregulated PLK1 and might be considered for entry into early phase clinical trials in pediatric patients.

Background: Medulloblastoma is a leading cause of childhood cancer-related deaths. Current aggressive treatments frequently lead to cognitive and neurological disabilities in survivors. Novel targeted therapies are required to improve outcome in high-risk medulloblastoma patients and quality of life of survivors. Targeting enzymes controlling epigenetic alterations is a promising approach recently bolstered by the identification of mutations in histone demethylating enzymes in medulloblastoma sequencing efforts. Hypomethylation of lysine 4 in histone 3 (H3K4) is also associated with a dismal prognosis for medulloblastoma patients. Functional characterization of important epigenetic key regulators is urgently needed., Results: We examined the role of the H3K4 modifying enzyme, KDM1A, in medulloblastoma, an enzyme also associated with malignant progression in the closely related tumor, neuroblastoma. Re-analysis of gene expression data and immunohistochemistry of tissue microarrays of human medulloblastomas showed strong KDM1A overexpression in the majority of tumors throughout all molecular subgroups. Interestingly, KDM1A knockdown in medulloblastoma cell lines not only induced apoptosis and suppressed proliferation, but also impaired migratory capacity. Further analyses revealed bone morphogenetic protein 2 (BMP2) as a major KDM1A target gene. BMP2 is known to be involved in development and differentiation of granule neuron precursor cells (GNCPs), one potential cell of origin for medulloblastoma. Treating medulloblastoma cells with the specific KDM1A inhibitor, NCL-1, significantly inhibited growth in vitro., Conclusion: We provide the first evidence that a histone demethylase is functionally involved in the regulation of the malignant phenotype of medulloblastoma cells, and lay a foundation for future evaluation of KDM1A-inihibiting therapies in combating medulloblastoma.